While the power of next-generation sequencing technologies to inform and guide malaria control programs has become broadly recognized, the integration of genomic data for operational incorporation into malaria surveillance remains a challenge in most countries where malaria is endemic. The main obstacles include limited infrastructure, limited access to high-throughput sequencing facilities, and the need for local capacity to run an in-country analysis of genomes at a large-enough scale to be informative for surveillance.

Targeted selection-based-genome-editing approaches in budding yeast have enabled many fundamental discoveries and continue to be used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette led to reduced expression and function for the adjacent gene, MRP51, despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all phenotypes we detected in cells deleted for DBP1. This behavior resembled the previously observed 'neighboring gene effect' (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). We demonstrate that divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests its consideration in the design and interpretation of experiments using integrated expression cassettes in other eukaryotes.

BackgroundMalaria molecular surveillance has great potential to support local national malaria control programs (NMCPs) to inform policy for malaria control and elimination. Molecular markers associated with drug resistance are good predictors of treatment responses. In addition, molecular detection of deletions in hrp2 and hrp3 genes are indicative of potential failure of HRP2-based rapid diagnostic tests. However, there is an urgent need for feasible, cost-effective and fast molecular surveillance tools that NMCPs can implement.MethodsHere we present a new 3-day workflow for targeted resequencing of markers in 13 resistance-associated genes, hrp2&3, a country-specific 28 SNP-barcode for population genetic analysis, and ama1. The assay was applied to control isolates and retrospective samples collected between 2003-2018 in the Loreto region (n = 254) in Peru. Pf AmpliSeq libraries were prepared using a multiplex PCR simultaneously amplifying a high number of targets from dried blood spots and sequenced at high coverage (median 1336, range 20-43795).ResultsThere was no evidence suggesting the emergence of artemisinin resistance in Peru. However, alleles in ubp1 and coronin contributed to recent genetic differentiation of the parasite population. After 2008, predominant parasite lineages in Peru are resistant to sulfadoxine-pyrimethamine (sextuple dhfr/dhps mutant) and chloroquine (SVMNT in crt and NDFCDY in mdr1) and can escape HRP2 based RDTs.ConclusionsThese findings indicate a parasite population under drug pressure, and demonstrates the added value of molecular surveillance systems and offers a highly multiplexed surveillance tool. The targets in the assay can be easily adjusted to suit the needs of other settings.FundingThis work was funded by the Belgium Development Cooperation (DGD) under the Framework Agreement Program between DGD and ITM (FA4 Peru, 2017-2021) and the sample collections in 2018 were supported by VLIR-UOS (project PE2018TEA470A102; University of Antwerp). Funding for the sample collections lead by the U.S. Naval Medical Research Unit 6 (NAMRU-6) in 2011 and 2012 was provided by the Armed Forces Health Surveillance Division (AFHSD) and its Global Emerging Infections Surveillance and Response (GEIS) Section (P0144_20_N6_01, 2020-2021).

Precision genome-editing approaches have long been available in budding yeast, enabling introduction of gene deletions, epitope tag fusions, and promoter swaps through a selection-based strategy. Such approaches allow loci to be modified without disruption of coding or regulatory sequences of neighboring genes. Use of this approach to delete DBP1 however, led to silencing of expression and the resultant loss of function for the neighboring gene MRP51. We found that insertion of a resistance cassette to delete DBP1, drove a 5’ extended alternative transcript for MRP51 which dampened Mrp51 protein synthesis. Misregulation of MRP51 occurred through an integrated transcriptional and translational repressive long undecoded transcript isoform (LUTI)-based mechanism that was recently shown to naturally regulate gene expression in yeast and other organisms. Cassette-induced MRP51 repression drove all mutant phenotypes we detected in cells deleted for DBP1. Selection cassette-mediated aberrant transcription events are not specific to this locus or a unique cassette but can be prevented by insertion of transcription insulators flanking the cassette. Our study suggests the existence of confounding off-target mutant phenotypes resulting from misregulated neighboring loci following genome edits in yeast. Furthermore, features of LUTI-based regulation are broadly conserved to eukaryotic organisms which indicates the potential that similar misregulation could be unnoticed in other edited organisms as well.

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